The cytoskeleton is essential to many aspects of life, cell division and motility being prime examples. The overarching goal of this proposal is to understand the roles of two polarity factors, Spire (Spir) and Cappuccino (Capu), in regulating the cytoskeleton and establishing the body axes in early Drosophila development. Spir and Capu are distinct types of actin nucleators, factors that build new actin filaments de novo. Mutations in either gene cause female sterility due to polarity and cytoskeletal defects, which indicates that they are involved in the same biological pathway. The most notable cytoskeletal defect is the loss of an actin mesh that traverses the Drosophila oocyte up until a dynamic process called cytoplasmic streaming begins. Interestingly, the Capu homolog, Fmn-2, was recently found to build an essential actin structure in mammalian oocytes suggesting functional conservation of Capu and perhaps Spir. In the proposed work, we will test three models of how Spir and Capu build actin structures and establish polarity in Drosophila oocytes: 1) the co-nucleation model, in which Spir and Capu nucleate collaboratively to build the actin mesh; 2) the synergy model, in which Spir is not a nucleator but enhances nucleation by Capu indirectly; 3) the crosslinking model, in which Spir and Capu regulate streaming by crosslinking the actin and microtubule cytoskeletons, as opposed to nucleating a mesh. The first and second models will be distinguished by using fluorescence microscopy, electron microscopy and other biochemical approaches to study the effects of Spir on actin filament dynamics. These experiments will allow direct observation of actin filaments to determine whether Spir primarily nucleates new filaments, severs existing filaments or alters filament dynamics. The first and third models will be distinguished using knowledge gained from in vitro experiments to design rational mutations in Spir and Capu. The mutations will be introduced into Drosophila to determine which activities of these two proteins are essential for their activities in vivo. Additional experiments will be performed to determine how the activities of these proteins are controlled. Distinguishing between these models will lead to a mechanistic understanding of two proteins, conserved throughout metazoan species. It will advance our knowledge of the cytoskeleton and how it is controlled. Given the co-existence of this pair of proteins in polar cells, including neurons and epithelial cells in mammals, it has been proposed that their role as polarity factors in Drosophila is conserved. Thus we anticipate that what is learned about Spir and Capu in Drosophila oogenesis will be applicable to our understanding of the cytoskeleton, cell polarity, fertility, development and health in many animals, ranging from Drosophila to humans. ) )